Antigenic epitopes with LYM-1 reactivity and uses thereof

ABSTRACT

This invention provides novel peptide epitopes recognized by the non-Hodgkin&#39;s B cell lymphoma reactive Lym-1 antibody. These novel peptide epitopes are capable of generating antibodies directed against Lym-1 peptide epitope expressing B-NHL cells. This invention is also directed to the treatment of B-NHL.

FIELD OF THE INVENTION

This invention has identified novel peptide epitopes recognized by the Bcell lymphoma-reactive Lym-1 antibody. Because Lym-1 specifically reactswith non-Hodgkin's B cell lymphoma cells, the invention provides animproved, accurate means to identify cancer patients potentiallyresponsive to Lym-1 antibody used as a cytotoxic therapeutic reagent.The invention also provides methods of generating antibodies directedagainst non-Hodgkin's B cell lymphoma cells which can be used in thetreatment of non-Hodgkin's B cell lymphoma.

INTRODUCTION

Low grade B-cell non-Hodgkin's lymphomas (B-NHL) represent a markedlyheterogeneous group of lymphoproliferative disorders (Gaidano (1997)Leuk. Lymphoma 26 Suppl. 1:107-113; Gandini (1996) Cancer GenetCytogenet. 86:120-123). A widely used treatment for these lymphomasinvolves administration of a B-NHL-specific antibody, called Lym-1.Lym-1 is a murine IgG_(2a) monoclonal antibody. When conjugated tocytotoxic agents, Lym-1 targets and kills B-NHL lymphoma cells (see,e.g., Rose (1996) Cancer Immunol. Immunother. 43:26-30; Epstein (1987)Cancer Res. 47:830-840). Lym-1 has been radiolabeled with ¹³¹I (see,e.g., DeNardo (1997) Cancer 80:2706-2711) and conjugated to the ribosomeinactivating protein gelonin (see, e.g., O'Boyle (1995) J. Immunother.Emphasis Tumor Immunol. 18:221-230). However, these reagents areinherently toxic, and not all B-NHL patients have Lym-1 reactive cancercells. Thus, there is a need for a means to identify which patients willbe responsive to such immunotherapy. While Lym-1 binding is associatedwith the expression of HLA DR10 by a patient, unfortunately, the absenceof DR10, does not consistently correlate with the presence or absence ofthe Lym-1 reactive epitope. In another words, Lym-1 can react with HLADR molecules other than DR10. Thus, there is a great need for a means toquickly, efficiently, and accurately determine the presence of a Lym-1reactive epitope in a B-NHL cancer patient. The present invention, whichfor the first time identifies Lym-1 reactive peptide epitopes, fulfillsthese and other needs.

Typically, no immune response is generated by the cancer patient againstB-NHL cells. However, based on studies with other tumor specificantigens (e.g., PSA antigen in prostate cancer), identification of animmunogenic peptide, followed by its administration with adjuvant, canelicit a tumor-specific immune response (see, e.g., Correale (1998) J.Immunol. 161:3186-3194). See also, Gjertsen (1998) Vox Sang. 74 Suppl2:489-495, who uses an immunogenic peptide from a carcinogenic, mutantras polypeptide to generate an immune response to pancreatic- andcolorectal adenocarcinomas. The present invention, by identifying Lym-1reactive epitopes on B-NHL cells, provides such a therapeuticimmunogenic peptide.

SUMMARY OF THE INVENTION

The invention for the first time provides a composition comprising anisolated or recombinant peptide comprising a subsequence of a Class IImajor histocompatibility molecule that generates an immune response to anon-Hodgkin's B cell lymphoma cell. The peptide of the invention has astructure comprisingR₁-R₂-R₃-R₄-R₅-R₆-R₇-R₈-R₉-R₁₀-R₁₁-R₁₂-R₁₃-R₁₄-R₁₅-R₁₆, wherein R₁ isGln, Lys, or Arg; R₂ is Arg; R₃ and R₄ are members independentlyselected from the group consisting of all amino acids; R₅ is Ala, Glu,Asp, Val, Leu or Ile; R₆ and R₇ are members independently selected fromthe group consisting of all amino acids; R₈ is Thr; R₉, R₁₀, R₁₁, R₁₂,R₁₃, R₁₄, and R₁₅ are members independently selected from the groupconsisting of all amino acids; and, R₁₆ is Val.

In one embodiment, the peptide of the invention has a structure whereinR₁ is Gln, Lys, or Arg; R₂ is Arg; R₃ is Arg; R₄ is selected from thegroup consisting of all amino acids; R₅ is Ala; R₆ and R₇ are membersindependently selected from the group consisting of all amino acids; R₈is Thr; R₉ is selected from the group consisting of all amino acids; R₁₀is Cys; R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅ are members independently selectedfrom the group consisting of all amino acids; and, R₁₆ is Val (SEQ IDNO: 1). In a preferred embodiment, the immunogenic peptide comprises astructure wherein R₁ is Gln, Lys, or Arg; R₂ is Arg; R₃ is Arg; R₄ isAla; R₅ is Ala; R₆ is Val; R₇ is Asp; R₈ is Thr; R_(is Tyr; R) ₁₀ isCys; R₁₁ is Arg; R₁₂ is His; R₁₃ is Asn; R₁₄ is Tyr; R₁₅ is Gly, and R₁₆is Val (SEQ ID NO: 2).

In alternative embodiments, the composition further comprises apharmaceutically acceptable excipient and an adjuvant.

The composition of the invention can generate an immune response to anon-Hodgkin's lymphoma cell (B-NHL), including, e.g., a B-cell chroniclymphocytic leukemia/small lymphocytic lymphoma (B-CCL/SLL) cell, alymphoplasmacytoid lymphoma (LPL) cell, a follicular lymphoma (FL) cell,a mucosa-associated lymphoid tissue lymphoma (MALTL) cell, a spleniclymphoma with villous lymphocytes (SLVL) cell and a mantle cell lymphomacell.

The invention also provides a method for detecting a nucleic acid in abiological sample, wherein the nucleic acid encodes a peptide capable ofspecifically binding to a Lym-1 antibody. The method of the inventioncomprises contacting the sample with an oligonucleotide primer paircapable of amplifying a subsequence of an MHC nucleic acid, whichsubsequence encodes a polypeptide comprising a peptide of the invention,as described above; amplifying the nucleic acid; and, detecting theamplified nucleic acid. In alternative embodiments, the MHC gene isHLA-DR 10 and the subsequence encodes a peptide wherein R₁ is Gln, Lys,or Arg; R₂ is Arg; R₃ is Arg; R₄ is Ala; R₅ is Ala; R₆ is Val; R₇ isAsp; R₈ is Thr; R₉ is Tyr; R₁₀ is Cys; R₁₁ is Arg; R₁₂ is His; R₁₃ isAsn; R₁₄ is Tyr; R₁₅ is Gly, and R₁₆ is Val (SEQ ID NO: 2).

In the methods, the biological sample can comprise a B cell, orspecifically, a B lymphocytic non-Hodgkin's lymphoma cell (B-NHL). TheB-NHL cell can be a B-cell chronic lymphocytic leukemia/smalllymphocytic lymphoma (B-CCL/SLL) cell, a lymphoplasmacytoid lymphoma(LPL) cell, a follicular lymphoma (FL) cell, a mucosa-associatedlymphoid tissue lymphoma (MALTL) cell, a splenic lymphoma with villouslymphocytes (SLVL) cell and a mantle cell lymphoma cell. In alternativeembodiments the biological sample can be a body fluid sample or a biopsysample; and the body fluid sample can be a blood sample.

The invention further provides a kit for detecting a nucleic acid in abiological sample, wherein the nucleic acid encodes a peptide capable ofspecifically binding to a Lym-1 antibody. The kit comprises anoligonucleotide primer pair capable of amplifying a subsequence of anMHC gene or gene product, which subsequence encodes a polypeptidecomprising a peptide of the invention. In alternative embodiments, theMHC gene can be HLA-DR 10; and, the peptide can comprise a structurewherein R₁ is Gln, Lys, or Arg; R₂ is Arg; R₃ is Arg; R₄ is Ala; R₅ isAla; R₆ is Val; R₇ is Asp; R₈ is Thr; R₉ is Tyr; R₁₀ is Cys; R₁₁ is Arg;R₁₂ is His; R₁₃ is Asn; R₁₄ is Tyr; R₁₅ is Gly, and R₁₆ is Val (SEQ IDNO: 2).

In another embodiment, the kit of the invention can further comprise aninstructional material teaching a use of the kit, wherein theinstructional material indicates that the kit is used for the detectionof nucleic acid encoding a peptide reactive with a Lym-1 antibody andthat the polypeptide is associated with non-Hodgkin's B cell lymphomas.

The invention also provides a method for detecting an antibody reactivewith a non-Hodgkin's B cell lymphoma (B-NHL) cell. The method comprisescontacting a sample, which can be a biological sample, with acomposition of the invention under immunologically reactive conditions,and then detecting whether an antibody has specifically bound to thecomposition. In one embodiment, the composition comprises a peptidehaving a structure wherein R₁ is Gln, Lys, or Arg; R₂ is Arg; R₃ is Arg;R₄ is Ala; R₅ is Ala; R₆ is Val; R₇ is Asp; R₈ is Thr; R₉ is Tyr; R₁₀ isCys; R₁₁ is Arg; R₁₂ is His; R₁₃ is Asn; R₁₄ is Tyr; R₁₅ is Gly, and R₁₆is Val (SEQ ID NO: 2). In various embodiments of this method, theantibody is generated by a recombinant nucleic acid library, therecombinant nucleic acid is a phage display library, and the compositionis fixed to a solid surface.

The invention further provides a method for generating an antibodyreactive with a non-Hodgkin's B cell lymphoma (B-NHL) cell. The methodcomprises administering an immunogenically effective amount of acomposition of the invention to a mammal. The composition can comprise apeptide having a structure wherein R₁ is Gln, Lys, or Arg; R₂ is Arg; R₃is Arg; R₄ is Ala; R₅ is Ala; R₆ is Val; R₇ is Asp; R₈ is Thr; R₉ isTyr; R₁₀ is Cys; R₁₁ is Arg; R₁₂ is His; R₁₃ is Asn; R₁₄ is Tyr; R₁₅ isGly, and R₁₆ is Val (SEQ ID NO: 2). The B-NHL cell can be a B-cellchronic lymphocytic leukemia/small lymphocytic lymphoma (B-CCL/SLL)cell, a lymphoplasmacytoid lymphoma (LPL) cell, a follicular lymphoma(FL) cell, a mucosa-associated lymphoid tissue lymphoma (MALTL) cell, asplenic lymphoma with villous lymphocytes (SLVL) cell and a mantle celllymphoma cell.

The invention provides an immunogenic composition capable of elicitingan immunogenic response directed to a polypeptide epitope, wherein theepitope comprises an amino acid sequence having a structure comprisingR₁-R₂-R₃-R₄-R₅-R₆-R₇-R₈-R₉-R₁₀-R₁₁-R₁₂-R₁₃-R₁₄-R₁₅-R₁₆, wherein R₁ isGln, Lys, or Arg; R₂ is Arg; R₃ and R₄ are members independentlyselected from the group consisting of all amino acids; R₅ is Ala, Glu,Asp, Val, Leu or Ile; R₆ and R₇ are members independently selected fromthe group consisting of all amino acids; R₈ is Thr; R₉, R₁₀, R₁₁, R₁₂,R₁₃, R₁₄, and R₁₅ are members independently selected from the groupconsisting of all amino acids; and, R₁₆ is Val. In one embodiment, theepitope comprises a sequence wherein R₁ is Gln, Lys, or Arg; R₂ is Arg;R₃ is Arg; R₄ is Ala; R₅ is Ala; R₆ is Val; R₇ is Asp; R₈ is Thr; R₉ isTyr; R₁₀ is Cys; R₁₁ is Arg; R₁₂ is His; R₁₃ is Asn; R₁₄ is Tyr; R₁₅ isGly, and R₁₆ is Val (SEQ ID NO: 2). The immunogenic response cangenerate antibodies (i.e., a humoral response) specific for thepolypeptide epitope. Alternatively, the immunogenic response cangenerate an epitope specific cellular response.

The invention further provides a method of inducing an immunogenicresponse directed to a polypeptide epitope, comprising administering animmunogenically effective amount of a composition comprising apolypeptide epitope to a mammal, wherein the epitope comprises an aminoacid sequence having a structure comprisingR₁-R₂-R₃-R₄-R₅-R₆-R₇-R₈-R₉-R₁₀-R₁₁-R₁₂-R₁₃-R₁₄-R₁₅-R₁₆, wherein R₁ isGln, Lys Arg; R₂ is Arg; R₃ and R₄ are members independently selectedfrom the group consisting of all amino acids; R₅ is Ala, Glu, Asp, Val,Leu or Ile; R₆ and R₇ are members independently selected from the groupconsisting of all amino acids; R₈ is Thr; R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄,and R₁₅ are members independently selected from the group consisting ofall amino acids; and, R₁₆ is Val. In one embodiment, the epitopecomprises an amino acid sequence having a structure wherein R₁ is Gln,Lys, or Arg; R₂ is Arg; R₃ is Arg; R₄ is Ala; R₅ is Ala; R₆ is Val; R₇is Asp; R₈ is Thr; R₉ is Tyr; R₁₀ is Cys; R₁₁ is Arg; R₁₂ is His; R₁₃ isAsn; R₁₄ is Tyr; R₁₅ is Gly, and R₁₆ is Val (SEQ ID NO: 2). Theimmunogenic response can generate antibodies (i.e., a humoral response)specific for the polypeptide epitope. Alternatively, the immunogenicresponse can generate an epitope specific cellular response. In variousembodiments, the method involves administering the immunogeniccomposition to a human, a mouse or a rabbit.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification, the figures and claims.

All publications, patents and patent applications cited herein arehereby expressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic illustration of the critical Lym-1 bindingresidues on the HLA Class II allele, DR1, as described in furtherdetail, below.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based at least in part on the discovery of peptideepitopes recognized by the non-Hodgkin's B cell lymphoma(B-NHL)-reactive Lym-1 antibody. Because Lym-1 specifically reacts withB-NHL cells, the invention provides an improved, accurate means toidentify which B-NHL patients will be responsive to a therapeutic Lym-1antibody. The invention, by identifying these novel Lym-1 epitopes, alsoprovides methods of generating an immune response, particularlyantibodies, directed against B-NHL cells bearing the Lym-1 epitope.

In one embodiment, these peptides are incorporated into immunogeniccompositions and administered to mammals, e.g., non-human animals, togenerate antibodies with high affinity for B-NHL cells expressing Lym-1epitopes. These antibodies are reactive with all Lym-1 epitopeexpressing cancer cells, including B-cell chronic lymphocyticleukemia/small lymphocytic lymphoma (B-CCL/SLL), lymphoplasmacytoidlymphomas (LPL), follicular lymphomas (FL), mucosa-associated lymphoidtissue lymphomas (MALTL), splenic lymphoma with villous lymphocytes(SLVL), and mantle cell lymphomas. In another embodiment, theimmunogenic compositions of the invention are administeredtherapeutically to cancer patients with a B cell lymphoma expressing aLym-1 epitope.

The invention further provides means to identify patients who will beresponsive to Lym-1 antibody therapy. Because the novel peptides of theinvention comprise the peptide epitope recognized by Lym-1, theinvention provides an improved, accurate means to identify cancerpatients potentially responsive to Lym-1 antibody used as a cytotoxictherapeutic reagent. The polymerase reverse transcriptase (PCR)-basedmethods and kits are especially needed for pre-screening B-NHL patientsbefore such Lym-1 therapy.

Definitions

To facilitate understanding the invention, and to provide additionalguidance to one of skill in the practice of the invention, a number ofterms are defined below. Unless defined otherwise, all technical andscientific terms used herein have the meaning commonly understood by aperson skilled in the art to which this invention belongs. The followingreferences provide one of skill with a general definition of many of theterms used in this invention: Singleton et al., Dictionary ofMicrobiology and Molecular Biology (2d ed. 1994); The CambridgeDictionary of Science and Technology (Walker ed., 1988); and Hale &Marham, The Harper Collins Dictionary of Biology (1991). Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,preferred methods and materials are described. As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

The term “amino acid” as used herein refers to all naturally occurringamino acids and altered (i.e., a chemically modified naturally occurringamino acid) and synthetic, i.e., “mimetic” (defined below) amino acidresidues which have substantially the same structure and function.

The term “amplifying” as used herein incorporates its common usage andrefers to the use of any suitable amplification methodology forgenerating recombinant nucleic acid, as described in detail, below. Forexample, the invention provides methods and reagents (e.g.,oligonucleotide PCR primer pairs) for amplifying nucleic acid encodingthe novel Lym-1 epitope of the invention.

The term “antibody” refers to a peptide or polypeptide substantiallyencoded by an immunoglobulin gene or immunoglobulin genes, or fragmentsthereof, capable of specifically binding an epitope, see, e.g.Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press,N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-73; Yarmush(1992) J. Biochem. Biophys. Methods 25:85-97. One of skill willappreciate that antibody fragments may be isolated or synthesized denovo either chemically or by utilizing recombinant DNA methodology. Theterm antibody also includes “chimeric” antibodies either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies. Typically, such chimeric antibodies are“humanized antibodies,” i.e., where the epitope binding site isgenerated from an immunized mammal, such as a mouse, and the structuralframework is human. Immunoglobulins can also be generated using phagedisplay libraries, and variations thereof, as described below.

The term “capable of generating an immune response to a non-Hodgkin's Bcell lymphoma cell” means that the composition of the invention, whenadministered in an appropriate amount (the “immunogenically effectiveamount,” defined and discussed, infra), either alone or in combinationwith another substance (such as, e.g., an adjuvant) or an immunologiccarrier (defined infra), can elicit a humoral or cellularantigen-specific immune response.

The term “Lym-1 antibody” refers to a murine IgG_(2a) monoclonalantibody that selectively binds an antigen, typically an HLA DR molecule(defined below), which is highly expressed on the surface of most B-NHLlymphomas (see, e.g., Rose (1996) Cancer Immunol. Immunother. 43:26-30;Epstein (1987) Cancer Res. 47:830-840). Lym-1 is used as an anti-cancerpharmaceutical after radiolabeling with a cytotoxic agent, such as,e.g., ¹³¹I (DeNardo (1997) supra), copper-67 (Denardo (1998) AnticancerRes. 18(4B):2779-88; Deshpande (1988) J. Nucl. Med. 29:217-225) orconjugation to the ribosome inactivating protein gelonin (O'Boyle (1995)supra; Boyle (1996) J. of Immunol. 18:221-230). The Lym-1 antigen isbelieved to be a variant form of HLA class II DR which is highlyexpressed on the surface of malignant B cells, but is found only at lowlevels in normal cells (see, e.g., Wurflein (1998) Cancer Res.58:3051-8). That portion of the antigen that contacts the antibody isreferred to as the “epitope.” The present invention has, for the firsttime, defined the peptide which is the Lym-1 antibody reactive epitope.

The terms peptide and polypeptide include naturally occurring aminoacids linked by peptide bonds and all “conservative variants,” “analogs”and “mimetics” (defined below) which have substantially the samestructure and function. Thus, the peptide and polypeptides of theinvention comprising the Lym-1-reactive epitope include all conservativevariants and mimetics that retain the ability to specifically bind tothe Lym-1 antibody. A peptide or polypeptide with one or more of such“conservative substitutions” is also within the scope of the inventionif it, as part of a composition, when administered in an immunogenicallyeffective amount to a mammal, is capable of generating an antibodyreactive with a B-NHL cell expressing the Lym-1 epitope. These includeconservatively modified variations of the epitope's amino acid sequence,i.e., amino acid substitutions, additions or deletions of residues inthe sequences provided herein, or substitution of amino acids with otheramino acid residues or mimetics having similar properties (e.g., acidic,basic, positively or negatively charged, polar or non-polar, etc.) suchthat the substitutions of even critical amino acids does notsubstantially alter structure and activity. Conservative substitutiontables providing functionally similar amino acids are well known in theart. For example, one exemplary guideline to select conservativesubstitutions includes (original residue followed by exemplarysubstitution): ala/gly or ser; arg/lys; asn/gln or his; asp/glu;cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln; ile/leu orval; leu/ile or val; lys/arg or gln or glu; met/leu or tyr or ile;phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe;val/ile or leu. An alternative exemplary guideline uses the followingsix groups, each containing amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); (see also, e.g., Creighton (1984) Proteins, W. H.Freeman and Company; Schulz and Schimer (1979) Principles of ProteinStructure, Springer-Verlag). One of skill in the art will appreciatethat the above-identified substitutions are not the only possibleconservative substitutions. For example, for some purposes, one mayregard all charged amino acids as conservative substitutions for eachother whether they are positive or negative. In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids in an encodedsequence can also be considered “conservatively modified variations.”Routine experimentation will determine whether a conservative variant iswithin the scope of the invention, i.e., that its structure and/orfunction (i.e., its ability to generate an antibody reactive with aLym-1 epitope-expressing B-NHL cell) is not substantially altered. Anexemplary means to test for such reactivity is described herein.

The terms amino acid “mimetic” and “peptidomimetic” refer to a syntheticchemical compound which has substantially the same structural andfunctional characteristics of the Lym-1 reactive epitope of theinvention. The mimetic can be either entirely composed of synthetic,non-natural analogues of amino acids, or, is a chimeric molecule ofpartly natural peptide amino acids and partly non-natural analogs ofamino acids. The mimetic can also incorporate any amount of naturalamino acid conservative substitutions as long as such substitutions alsodo not substantially alter the mimetic's structure and/or activity(i.e., capability of generating an antibody reactive with a Lym-1epitope-expressing B-NHL cell). As with peptides and polypeptides of theinvention which are conservative variants, routine experimentation willdetermine whether a mimetic is within the scope of the invention, andexemplary means to screen for such binding is described herein. Aminoacid mimetic containing compositions can have any combination ofnonnatural structural components, which are typically from threestructural groups: a) residue linkage groups other than the naturalamide bond (“peptide bond”) linkages; b) non-natural residues in placeof naturally occurring amino acid residues; or c) residues which inducesecondary structural mimicry, ie., to induce or stabilize a secondarystructure, e.g., a beta turn, gamma turn, beta sheet, alpha helixconformation, and the like (see discussion, below, on Lym-1 reactiveepitope secondary structure). A peptide can be characterized as amimetic when all or some of its residues are joined by chemical meansother than natural peptide bonds. Individual peptidomimetic residues canbe joined by peptide bonds, other chemical bonds or coupling means, suchas, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctionalmaleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC). Linking groups that can be analternative to the traditional amide bond (“peptide bond”) linkagesinclude, e.g., ketomethylene (e.g., —C(═O)—CH₂— for —C(═O)—NH—),aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether (CH₂—O),thioether (CH₂—S), tetrazole (CN₄—), thiazole, retroamide, thioamide, orester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of AminoAcids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide BackboneModifications,” Marcell Dekker, NY). A polypeptide can also becharacterized as a mimetic by containing all or some non-naturalresidues in place of naturally occurring amino acid residues. Nonnaturalresidues are well described in the scientific and patent literature; afew exemplary nonnatural compositions useful as mimetics of naturalamino acid residues and guidelines are described below. Mimetics ofaromatic amino acids can be generated by replacing by, e.g., D- orL-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- orL-1, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenyl-alanine; D- or L-p-biphenylphenylalanine; K- orL-p-methoxy-biphenyl-phenylalanine; D- or L-2-indole(alkyl)alanines;and, D- or L-alkylainines, where alkyl can be substituted orunsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl,iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromaticrings of a nonnatural amino acid include, e.g., thiazolyl, thiophenyl,pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridylaromatic rings. Mimetics of acidic amino acids can be generated bysubstitution by, e.g., non-carboxylate amino acids while maintaining anegative charge; (phosphono)alanine; sulfated threonine. Carboxyl sidegroups (e.g., aspartyl or glutamyl) can also be selectively modified byreaction with carbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl)carbodiimide. Aspartyl or glutamylcan also be converted to asparaginyl and glutaminyl residues by reactionwith ammonium ions. Mimetics of basic amino acids can be generated bysubstitution with, e.g., (in addition to lysine and arginine) the aminoacids omithine, citrulline, or (guanidino)-acetic acid, or(guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrilederivative (e.g., containing the CN-moiety in place of COOH) can besubstituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups. A component of a naturalpolypeptide (e.g., the peptide of the invention) can also be replaced byan amino acid (or peptidomimetic residue) of the opposite chirality.Thus, any amino acid naturally occurring in the L-configuration (whichcan also be referred to as the R or S, depending upon the structure ofthe chemical entity) can be replaced with the amino acid of the samechemical structural type or a peptidomimetic, but of the oppositechirality, generally referred to as the D-amino acid, or, the R- orS-form.

The term “Major Histocompatibility Complex gene” or “MHC gene” is usedin its common usage and refers to genes and gene products encoding MajorHistocompatibility Complex (MHC) molecules. Human MHC molecules can alsobe referred to as “Human Leukocyte Antigens” or HLA. Class II DR MHCmolecules are heterodimers displayed on the cell surface of antigenprocessing/presenting cells (APCs), which include, e.g., human B cells.Cancer cells, e.g., B cell lymphomas, can also express DR molecules(see, e.g., Guy (1986) Br. J. Cancer 53:161-73; Rask (1991) Autoimmunity8:237-44; Jones (1997) Curr. Opin. Immunol. 9:75-9).

The term “subsequence of a Class II major histocompatibility molecule”refers to a polypeptide or peptide that has a sequence that is less thana naturally occurring full length sequence of a Class II MHCpolypeptide. For example, in alternative embodiments, the peptide of theinvention is at least about 16 residues in length, at least about 25residues in length, at least about 35 residues in length, at least about45 residues in length, at least about 55 residues in length, at leastabout 65 residues in length, at least about 75 residues in length, andat least about 85 residues in length.

An “immunogen” refers to a compound or composition comprising a peptide,polypeptide or protein which is “immunogenic” when administered in anappropriate amount (the “immunogenically effective amount”), i.e.,capable of eliciting, augmenting or boosting a cellular and/or humoralimmune response, either alone or in combination or linked or fused toanother substance (which can be administered at once or over severalintervals). An immunogenically effective amount can vary depending onwhether it is to be administered in intervals, versus a single dose. Animmunogenic composition can be, e.g., a peptide of at least about 5amino acids, a peptide of 10 amino acids in length, or a peptide of 16amino acids in length. For example, the peptide of the invention if atleast about 16 residues in length and is capable of generating anantibody specifically reactive with a Lym-1 epitope expressing B-NHLcell.

The immunogen can comprise a “carrier” polypeptide and a hapten, e.g., afusion protein or a carrier polypeptide fused or linked (chemically orotherwise) to a peptide epitope (discussed below). The immunogen can berecombinantly expressed in an immunization vector, which can be simplynaked DNA comprising the immunogen's coding sequence operably linked toa promoter. For example, the invention provides an immunogen capable ofgenerating an antibody specifically reactive with a Lym-1 epitopeexpressing B-NHL cell.

An “immunological carrier” or “carrier” in the immunological context (asopposed to a carrier which is a nonactive composition for the purpose offormulating, storing or carrying a pharmaceutical) is an compositionwhich, when linked, joined, chemically coupled or fused to a secondcomposition (e.g., protein, peptide, polysaccharide or the like) boostsor augments the cellular or humoral response to the composition. Anyphysiologic mechanism can be involved in this augmentation or boostingof the immune response. An immunogenic carrier is typically apolypeptide linked or fused to a second composition (the secondcomposition comprises the epitope/antigen intended to generate theimmune response, e.g., the Lym-1 reactive peptide epitope of theinvention). The carrier stimulates a cellular (T cell mediated) immuneresponse that boosts or augments the humoral (B cell mediated,antibody-generating) immune response to the epitope-containingcomposition of interest (e.g., a peptide of the invention). These secondcompositions can be “haptens,” which are typically defined as compoundsof low molecular weight that are not immunogenic by themselves, butthat, when coupled to carrier molecules, can elicit antibodies directedto epitopes on the hapten. For example, a peptide of the invention maybe a hapten which, when conjugated to a carrier, becomes sufficientlyimmunogenic to generate an immune response. Alternatively, a peptide ofthe invention can be linked to a carrier simply to facilitatemanipulation of the peptide in the generation of an immune response(see, e.g., Rondard (1997) Biochemistry 36:8962-8968).

The term “immunogenic response” refers to the generation of antibodiesand/or a cell-mediated immune response elicited by the introduction ofan immunogenic composition to a mammal.

As used herein, “isolated,” when referring to a molecule or composition,such as, for example, a peptide or nucleic acid, means that the moleculeor composition is separated from at least one other compound, such as aprotein, other nucleic acids (e.g., RNAs), or other contaminants withwhich it is associated in vivo or in its naturally occurring state.Thus, a polypeptide or nucleic acid is considered isolated when it hasbeen isolated from any other component with which it is naturallyassociated, e.g., cell membrane, as in a cell extract. An isolatedcomposition can, however, also be substantially pure. An isolatedcomposition can be in a homogeneous state and can be in a dry or anaqueous solution. Purity and homogeneity can be determined using avariety of analytical chemistry techniques such as, e.g., polyacrylamidegel electrophoresis (PAGE), high performance liquid chromatography(HPLC), and NMR spectroscopy.

The term “nucleic acid” or “nucleic acid sequence” refers to adeoxy-ribonucleotide or ribonucleotide oligonucleotide in either single-or double-stranded form. The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotideswhich have similar or improved binding (hybridization) properties, forthe purposes desired, e.g., amplification of nucleic acid encoding theLym-1 reactive peptide of the invention, as the reference nucleic acid.The term also encompasses nucleic-acid-like structures with syntheticbackbones, see e.g., Oligonucleotides and Analogues, a PracticalApproach, edited by F. Eckstein, IRL Press at Oxford University Press(1991); Antisense Strategies, Annals of the New York Academy ofSciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan(1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications(1993, CRC Press), WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl.Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698;Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156).

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a subject. The pharmaceutical compositions ofthis invention are formulations that comprise a pharmacologicallyeffective amount of a composition comprising a peptide capable ofgenerating an antibody specifically reactive to a Lym-1epitope-expressing B-NHL cell, and a pharmaceutically acceptablecarrier.

Nucleic Acids Encoding Lym-1 Reactive Peptides

This invention provides novel, immunogenic Lym-1-reactive peptides andnucleic acids encoding these peptides, including oligonucleotidescapable of amplifying this newly identified Lym-1 epitope. Synthetic andrecombinant forms of epitope-encoding nucleic acids are provided. Theinvention also provide means to express the epitope encoding nucleicacids of the invention to generate recombinant peptides andpolypeptides; which can be expressed in vitro or in vivo. The inventionprovides for a variety of means of expressing the nucleic acids andproteins of the invention, including expression cassettes, vectors, celllines, transgenic plants and animals, and the like.

The invention can be practiced in conjunction with any method orprotocol known in the art, which are well described in the scientificand patent literature, see e.g., Sambrook, ed., Molecular Cloning: ALaboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory,(1989) (“Sambrook”); Current Protocols in Molecular Biology, Ausubel,ed. John Wiley & Sons, Inc., New York (1997) (“Ausubel”); and,Laboratory Techniques in Biochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic AcidPreparation, Tijssen, ed. Elsevier, N.Y. (1993) (“Tijssen”).

Expression of Recombinant Lym-1 Reactive Peptides

The invention provides methods and reagents for recombinant expressionof the novel Lym-1 reactive epitopes of the invention. The nucleic acidsof the invention may be introduced into a genome or into the cytoplasmor a nucleus of a cell and expressed by a variety of conventionaltechniques, well described in the scientific and patent literature. Forexample, for expression of these nucleic acids, the inventionincorporates expression cassettes, vectors, transgenic plants andanimals, using constitutive and inducible transcriptional andtranslational cis-(e.g., promoters and enhancers) and trans-actingcontrol elements. The expression of nucleic acid coding for Lym-1reactive epitope can be achieved by operably linking the coding regionto a promoter incorporating the construct into an expression cassette(such as an expression vector), and introducing the resultant constructinto an in vitro reaction system or a suitable host cell or organism.See, e.g., Roberts (1987) Nature 328:731; Berger (1987) supra; Schneider(1995) Protein Expr. Purif. 6435:10; Sambrook, Tijssen or Ausubel.Product information from manufacturers of biological reagents andexperimental equipment also provide information regarding knownbiological methods. The promoters and vectors used in this invention canbe isolated from natural sources, obtained from such sources as ATCC orGenBank libraries, or prepared by synthetic or recombinant methods.

Amplification of Nucleic Acids Encoding Lym-1 Reactive Epitopes

The present invention provides oligonucleotide primers and probes thatcan hybridize specifically to nucleic acids to amplify sequencesencoding the Lym-1 reactive epitopes of the invention. Theseoligonucleotide primer reagents can be used to identify and quantifysuch sequences. Utilizing these reagents, the invention provides a kitand a method for detecting in a biological sample a nucleic acid thatencodes a peptide capable of specifically binding to a Lym-1 antibody.Illustrative PCR amplification methods for use in the kits and methodsof the invention are described below.

Oligonucleotides can be used to amplify the epitope-encoding nucleicacid of the invention using a variety of hybridization techniques andconditions. One of skill in the art will appreciate that, whateveramplification method is used, if a quantitative result is desired, caremust be taken to use a method that maintains or controls for therelative frequencies of the amplified nucleic acids. Suitableamplification methods include, but are not limited to: polymerase chainreaction, PCR (PCR Protocols, a Guide to Methods and Applications, ed.Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis,Academic Press, Inc., N.Y. (“Innis”)), ligase chain reaction (LCR) (Wu(1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer(1990) Gene 89:117); transcription amplification (Kwoh (1989) Proc.Natl. Acad. Sci. U.S.A. 86:1173); self-sustained sequence replication(Guatelli (1990) Proc. Natl. Acad. Sci. U.S.A., 87:1874); Q Betareplicase amplification (Smith (1997) J. Clin. Microbiol. 35:1477-1491,automated Q-beta replicase amplification (Burg (1996) Mol. Cell. Probes10:257-271); and other RNA polymerase mediated techniques (e.g., NASBA,Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol.152:307-316, Sambrook, and Ausubel, as well as Mullis (1987) U.S. Pat.Nos. 4,683,195 and 4,683,202; Arnheim (1990) C&EN 36-47; Lomell (1989)J. Clin. Chem. 35:1826; Van Brunt (1990) Biotechnology 8:291-294; Wu(1989) Gene 4:560; and Sooknanan (1995) Biotechnology 13:563-564.

In these amplification techniques, oligonucleotide primers are designedto be complementary to the two borders of the DNA region to be amplified(see, e.g., Innis). Thus, the invention provides oligonucleotide primerpairs with sequences comprising the Lym-1-reactive peptide epitopesequences of the invention. The invention also provides oligonucleotideprimer pairs capable of amplifying a subsequence of an MHC gene or geneproduct which contains the Lym-1-reactive peptide epitope sequences ofthe invention. Because the Lym-1 epitope is most commonly associatedwith the beta chain of the HLA DR10 allele, oligonucleotide primer pairscan include and can amplify subsequences of HLA DR10. Because thesequence of HLA DR10 is well known in the art, the skilled artisan canreadily design oligonucleotide (PCR) primer pairs capable of amplifyingsubsequences of MHC genes, e.g., DR10, comprising sequences coding forthe Lym-1 reactive epitopes of the invention. Furthermore, using publicdatabases, the skilled artisan can readily search for additional HLAalleles containing Lym-1-reactive peptide epitope sequences of theinvention and design appropriate oligonucleotide primer pairs capable ofamplifying these MHC gene subsequences. See, e.g., Gongora (1996) Hum.Immunol. 51:23-31; Gongora (1997) J. Immunol. 159:6044-51; Rose (1996)supra.

In one embodiment, the invention provides a method for detecting anucleic acid in a biological sample where the nucleic acid encodes apeptide capable of specifically binding to a Lym-1 antibody. In thesample, typically a cell (e.g. a B-NHL cell), is contacted with anoligonucleotide primer pair capable of amplifying the peptide-encodingnucleic acid (see, e.g., Bunce (1993) Hum. Immunol. 37:201-206). In apreferred embodiment, the PCR based methods and the kits of theinvention are used to identify B-NHL patients who will be response toLym-1 radiotherapy programs. This PCR based assay can be accomplishedwith a simple biological sample, i.e., from blood or saliva, that couldbe tested in parallel with, and eventually replace, the current need fora biopsy.

The invention's means to assess for Lym-1 reactivity provides a greatimprovement over the current criteria, which requires invasive biopsy orradioimaging with radioactive reagents. The present invention can bepracticed with only a simple blood test. The results presented herein(see Example 1, below) confirm that Lym-1 binds preferentially tolymphoblastoid B-NHL cells over normal PBLs, which may help explain theimpressive radio-immunodiagnostic and radio-immunotherapeutic resultsobtained so far in clinical trials of radiolabeled Lym-1.

Immunogenic Peptides and Polypeptides

This invention provides immunogenic peptides capable of generating animmune response, particularly antibodies, specifically directed to aLym-1 epitope expressing B-NHL cell. These peptides can be isolated fromnatural sources (e.g., as fragments of MHC Class II DR polypeptides),synthetic, or recombinantly generated polypeptides. Lym-1 epitopeexpressing peptides and proteins can be recombinantly expressed in vitroor in vivo. The peptides, polypeptides and complexes of the inventioncan be made and isolated using any method known in the art, and theinvention provides a few exemplary means for generating such proteins.

The peptides of the invention can also be synthesized, whole or in part,using chemical methods well known in the art (see e.g., Caruthers (1980)Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res.Symp. Ser. 225-232; Banga, A. K., Therapeutic Peptides and Proteins,Formulation, Processing and Delivery Systems (1995) Technomic PublishingCo., Lancaster, Pa. (“Banga”)). For example, peptide synthesis can beperformed using various solid-phase techniques (see e.g., Roberge (1995)Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) andautomated synthesis may be achieved, e.g., using the ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer.

The skilled artisan will recognize that individual synthetic residuesand polypeptides incorporating mimetics can be synthesized using avariety of procedures and methodologies, which are well described in thescientific and patent literature, e.g., Organic Syntheses CollectiveVolumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY. Polypeptidesincorporating mimetics can also be made using solid phase syntheticprocedures, as described, e.g., by Di Marchi, et al., U.S. Pat. No.5,422,426. Peptides and peptide mimetics of the invention can also besynthesized using combinatorial methodologies. Various techniques forgeneration of peptide and peptidomimetic libraries are well known, andinclude, e.g., multipin, tea bag, and split-couple-mix techniques; see,e.g., al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr.Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27;Ostresh (1996) Methods Enzymol. 267:220-234. Modified peptides of theinvention can be further produced by chemical modification methods, see,e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995)Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry33:7886-7896.

Peptides and polypeptides capable of generating an immune response toB-NHL cells can also be isolated from a natural sources, such as a cellline expressing the appropriate DR allele or a patient of theappropriate genotype (e.g., HLA DR10), using a variety of techniqueswell known in the art. Suitable HLA DR molecules can be isolated from Bor T cells which have been immortalized by transformation, e.g., as by Bcell transformation, utilizing techniques known in the art. See, e.g.,Rothenhausler (1990) Proc. Natl. Acad. Sci. U.S.A. 87:352-354; Gorga(1987) J. Biol. Chem. 262:16087-16094; Dommair (1989) Cold Spring HarborSymp. Quant. Biol. 54:409-416; Banga.

Fusion Proteins

Lym-1 reactive peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate Lym-1 epitope-reactive antibodies and antibody-expressing Bcells, and the like. Detection and purification facilitating domainsinclude, e.g., metal chelating peptides such as polyhistidine tracts andhistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequences such as Factor Xa or enterokinase(Invitrogen, San Diego Calif.) between the purification domain and Lym-1reactive peptide or polypeptide can be useful to facilitatepurification. For example, an expression vector can include anepitope-encoding nucleic acid sequence linked to six histidine residuesfollowed by a thioredoxin and an enterokinase cleavage site (see e.g.,Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr.Purif. 12:404-14). The histidine residues facilitate detection andpurification while the enterokinase cleavage site provides a means forpurifying the epitope from the remainder of the fusion protein.Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Peptide Secondary Structure

Previous work has shown that Lym-1 binds the isolated Class II DR10 betachain, but only when the intrachain disulfide bonds are intact (Rose(1996) supra). An affinity column using immobilized Lym-1 antibodyisolated the Lym-1 antigen from a Raji cell lysate. Characterization ofimmunoaffinity-purified Lym-1 antigen on Western blots demonstrated itto be the beta chain of HLA DR10. However, the Raji cell Lym-1 antigenis only recognized by Lym-1 antibody if the beta chain (intra-chain)disulfide bonds are intact. These results demonstrate that, at least insome cells, Lym-1 antibody binds a discontinuous epitope, i.e., binds toa conformational epitope formed by the folding of the polypeptideinduced by intra-molecular disulfide bonding.

As identified by the present invention, in the Raji cell DR10 Lym-1epitope, the intra-chain disulfide bond is formed between residues 15and 79, which brings a valine at residue 85 in relatively closeproximity to an arginine at residue 71; see the analogous threedimensional structure of a DR antigen in Brown (1993) Nature 364:33-39,in FIG. 1, from which the distances between the residues of the Lym-1epitope can be estimated.

This invention has, for the first time, determined the epitope for theLym-1 antibody on Class II DR10 polypeptides isR₁-R₂-R₃-R₄-R₅-R₆-R₇-R₈-R₉-R₁₀-R₁₁-R₁₂-R₁₃-R₁₄-R₁₅-R₁₆, where R₁ is Gln,Lys, or Arg; R₂ is Arg; R₃ is Arg; R₄ is Ala; R₅ is Ala; R₆ is Val; R₇is Asp; R₈ is Thr; R₉ is Tyr; R₁₀ is Cys; R₁₁ is Arg; R₁₂ is His; R₁₃ isAsn; R₁₄ is Tyr; R₁₅ is Gly, and R₁₆ is Val, or, simply: (Gln Lys, orArg)-Arg-Arg-Ala-Ala-Val-Asp-Thr-Tyr-Cys-Arg-His-Asn-Tyr-Gly-Val (SEQ IDNO: 2), which correspond to residues 70 to 85 on the DR10 polypeptide.Based on the secondary structure of DR molecules proposed by Brown(1993) supra, (see FIG. 1), in DR10, the valine at residue 85 inproximity to the arginine at residue 71 (because of the secondarystructure induced by the intrachain disulfide bond) corresponds toresidue R₁₆ and residue R₂, respectively, of the epitope of theinvention. Accordingly, in one embodiment, the invention provides apolypeptide or peptide composition which, in addition to having theabove-described amino acid sequence, has a secondary structure in thesame orientation with respect to each other as in the native molecule(estimated by the teaching of, e.g., the DR structure proposed by Brown(1993) supra). One of skill can readily test whether (and to whatdegree, i.e., what with what affinity) a peptide with a particularsecondary structure binds to Lym-1 antibody.

Peptidomimetics (i.e., completely or partially synthetic peptides) ofthe invention can be designed to incorporate secondary structure. Forexample, the design can ensure that, in the folded peptidomimetic, twoparticular residues are a predesignated distance from one another. Thus,a peptidomimetic of the invention can be designed to fold to haveresidues R₁₆ and residue R₂ the same distance apart as they would be inthe native Class II DR structure, as discussed above. Beta turns, betasheets, alpha helix structures, gamma turns, and the like can beincorporated into the peptidomimetic to effect this, or any other,design. For example, substitution of natural amino acid residues withD-amino acids; N-alpha-methyl amino acids; C-alpha-methyl amino acids;or dehydroamino acids within a peptide can induce or stabilize betaturns, gamma turns, beta sheets or alpha helix conformations. Beta turnmimetic structures have been described, e.g., by Nagai (1985) Tet. Lett.26:647-650; Feigl (1986) J. Amer. Chem. Soc. 108:181-182; Kahn (1988) J.Amer. Chem. Soc. 110:1638-1639; Kemp (1988) Tet. Lett. 29:5057-5060;Kahn (1988) J. Molec. Recognition 1:75-79. Beta sheet mimetic structureshave been described, e.g., by Smith (1992) J. Amer. Chem. Soc.114:10672-10674; Banerjee (1996) Biopolymers 39:769-777. Secondarystructures of polypeptides can be analyzed by, e.g., high-field 1H NMRor 2D NMR spectroscopy, see, e.g., Higgins (1997) J. Pept. Res.50:421-435. See also, Hruby (1997) Biopolymers 43:219-266, Balaji, etal., U.S. Pat. No. 5,612,895.

Generation of an Anti-B-NHL Immune Response

The Lym-1 reactive peptides and polypeptides of the invention can alsobe used to generate an immune response against Lym-1 epitope-expressingB-NHL cells. In one embodiment, they are used to generate antibodieswith equal or greater affinity than Lym-1 to epitope-expressing B-NHLcells. These antibodies can react with B-cell chronic lymphocyticleukemia/small lymphocytic lymphomas (B-CCL/SLL), lympho-plasmacytoidlymphomas (LPL), follicular lymphomas (FL), mucosa-associated lymphoidtissue lymphomas (MALTL), splenic lymphoma with villous lymphocytes(SLVL), or mantle cell lymphomas. Such antibodies can be generated invitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, or in vivo, e.g., using animals. Such recombinantanti-B-NHL antibodies can be further “humanized” for administration topatients with Lym-1 epitope-expressing B-NHL. Methods for makingchimeric, e.g., “humanized,” antibodies are well known in the art, seee.g., Wallace, et. al., U.S. Pat. No. 5,811,522; Leung, et. al., U.S.Pat. No. 5,789,554.

In one embodiment, the peptides of the invention are used as apharmaceutical, immunogenic composition to generate an anti-Lym-1expressing-B-NHL response in a human. The peptide can be conjugated toanother molecule or can be administered with an adjuvant. Alternatively,DNA encoding a polypeptide comprising the Lym-1 epitope of the inventioncan be administered as a pharmaceutical. The coding sequence is part ofan expression cassette or vector capable of expressing the immunogen invivo. (see, e.g., Katsumi (1994) Hum. Gene Ther. 5:1335-9).

The immunogenic compositions of the invention can generate an anti-Lym-1epitope specific cellular or a humoral (antibody) immune response.

The peptides of the invention can also be used to isolate, detect orquantify newly generated or existing anti-Lym-1 reactive antibodies, orto detect a cellular immune response.

Methods of producing polyclonal and monoclonal antibodies are known tothose of skill in the art and described in the scientific and patentliterature, see, e.g., Coligan, Current Protocols in Immunology,Wiley/Greene, NY (1991); Stites (eds.) Basic and Clinical Immunology(7th ed.) Lange Medical Publications, Los Altos, Calif. (“Stites”);Goding, Monoclonal Antibodies: Principles and Practice (2d ed.) AcademicPress, New York, N.Y. (1986); Kohler (1975) Nature 256:495; Harlow(1988) Antibodies, a Laboratory Manual, Cold Spring Harbor Publications,New York.

Such techniques include selection of Lym-1 reactive antibodies fromlibraries of recombinant antibodies displayed on phage (“phage displaylibraries”) or on cells. See, e.g., Huse (1989) Science 246:1275; Ward(1989) Nature 341:544; Hoogenboom (1997) Trends Biotechnol. 15:62-70;Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45. Recombinantantibodies can also be expressed by transient or stable expressionvectors in mammalian cells, as in Norderhaug (1997) J. Immunol. Methods204:77-87; Boder (1997) Nat. Biotechnol. 15:553-557.

Specific monoclonal and polyclonal antibodies and antisera will usuallybind with a K_(D) of at least about 1 μM, preferably at least about 0.1μM or better, and most preferably, 0.01 μM or better.

Immunoassays

The invention provides a composition which, when administered in animmunogenically effective amount to a mammal, is capable of generatingan antibody reactive with a Lym-1 epitope expressing B-NHL cell. Theinvention also provides a method for detecting an antibody reactive witha Lym-1 epitope expressing B-NHL cell using a peptide of the invention.Accordingly, the invention also provides reagents and methods using avariety of antibody based assays.

Immunological binding are well known in the art; see, e.g., U.S. Pat.Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168; Methods in CellBiology Vol. 37. Antibodies in Cell Biology, Asai, ed. Academic Press,Inc. New York (1993); Sambrook, Stites; Silzel (1998) Clin. Chem.44:2036-43; Rongen (1997) J. Immunol. Methods 204:105-133.; Hashida(1995) Biotechnol. Annu. Rev. 1:403-51; Bao (1997) J. Chromatogr. B.Biomed. Sci. Appl. 699:463-80; Self (1996) Curr. Opin. Biotechnol.7:60-5.

Formulation and Administration of Peptide Pharmaceutical Compositions

The invention also provides an immunogenic composition comprising apeptide epitope of the invention capable of eliciting an immunogenicresponse to that epitope. In one embodiment, the peptides andpolypeptides of the invention are administered with a pharmaceuticallyacceptable carrier(s) (excipient) to form the pharmacologicalcomposition.

Pharmaceutically acceptable carriers and formulations for peptides andpolypeptide are known to the skilled artisan and are described in detailin the scientific and patent literature, see e.g., the latest edition ofRemington's Pharmaceutical Science, Maack Publishing Company, Easton,Pa. (“Remington's”); Banga; Putney (1998) Nat. Biotechnol. 16:153-157;Patton (1998) Biotechniques 16:141-143; Edwards (1997) Science 276:1868-1871; Ho, et al., U.S. Pat. No. 5,780,431; Webb, et al., U.S. Pat.No. 5,770,700; Goulmy, et. al., U.S. Pat. No. 5,770,201.

The peptide and polypeptide immunogenic compositions used in the methodsof the invention can be delivered alone or as pharmaceuticalcompositions by any means known in the art, e.g., systemically,regionally, or locally; by intraarterial, intrathecal (IT), intravenous(IV), parenteral, intra-pleural cavity, topical, oral, or localadministration, as subcutaneous, intra-tracheal (e.g., by aerosol) ortransmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasalmucosa). Actual methods for delivering compositions will be known orapparent to those skilled in the art and are described in detail in thescientific and patent literature, see e.g., Remington's.

The pharmaceutical compositions can be administered by any immunizationprotocol and in a variety of unit dosage forms depending upon the methodof administration, whether an initial or booster dose is beingadministered, and the like. Immunogenic dosages for typical peptide andpolypeptide pharmaceutical compositions are well known to those of skillin the art. Such dosages are typically advisorial in nature and areadjusted depending on a variety of factors, e.g., the initial immuneresponse (e.g., antibody titers after initial immunization), theparticular therapeutic context, patient health and tolerance. The amountof peptide adequate to generate the desired immune response is definedas a “therapeutically effective dose.” The immunization and dosageschedule and amounts effective for this use, i.e., the “dosing regimen,”will depend upon a variety of factors, including the stage of the B-NHL,the severity of the disease or condition, the general state of thepatient's health, the patient's physical status, age, pharmaceuticalformulation and concentration of immunogen and/or adjuvant, and thelike. The dosage regimen also takes into consideration pharmacokinetics,i.e., the peptide pharmaceutical composition's rate of absorption,bioavailability, metabolism, clearance, and the like, see, e.g.,Remington.

The immunogenic composition can be repeated (“boosters”) in intervalsover weeks or months, depending on the patient's condition and initialimmune response.

Dosages can be determined empirically, by assessing the abatement oramelioration of symptoms, or by objective criteria, such analysis ofblood or histopathology specimens. Successful treatment can also bemonitored by histopathology. Thus, the immunogenic compositions of theinvention are administered to, e.g., arrest the progress, reduce theseverity, or prevent the recurrence of the Lym-1 epitope-expressingB-NHL.

The pharmaceutical compositions containing the peptide and complexes ofthe invention can be administered alone or in conjunction with othertherapeutic treatments. As noted above, a single or multipleadministrations (immunizations) of the compositions may be administereddepending on the dosage and frequency as required and tolerated by thepatient.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Identification of Peptide Epitopes Reactive With Anti-B-NHLAntibodies

The following example details the identification of a novel class ofpeptide epitopes reactive with the Lym-1 antibody.

The Lym-1 antibody was selected to initially identify an exemplary anepitope capable of generating an anti-B-NHL immune response because thismonoclonal antibody can react with most classes of B-NHL cells. As notedabove, Lym-1 is a murine IgG_(2a) monoclonal antibody (mAb) thatselectively binds an antigen which is highly expressed on the surface ofmost malignant human B-cells.

An HLA-DR10 allele present on a Raji cell line was the original antigenused to generate the Lym-1 murine monoclonal antibody. However, HLA-DR10is a relatively rare subtype found in less than 2% of the Americanpopulation, while greater than 80% of lymphoma patients screened forinclusion in Lym-1 radioimmunotherapy clinical trials were Lym-1reactive as determined by flow cytometry, immunoperoxidase staining andradioimmunodiagnostic imaging. This demonstrates that Lym-1 reacts withat least one epitope that is present in some HLA-DR10 alleles, but alsowith many other, but not all HLA-DR alleles. Thus, the data supports theexistence of several different Lym-1 reactive, and thus anti-B-NHLreactive, epitopes on many different DR beta chain alleles. The presentinvention, as described below, for the first time, identifies this novelclass of epitopes.

The basic strategy of these studies exploited the existence of over 100polymorphic variants of the human DR beta chains to design epitopemapping experiments (see, e.g., Erlich (1991) Hum. Immunol. 30:110-118).

Lymphoma Biopsy Studies

To structurally analyze polymorphic variants of the DR beta chainsreactive with Lym-1, Class II DR encoding nucleic acid amplified fromlymphoma cell lines and fresh cell isolates were analyzed. Lym-1reactive lymphoma biopsies was used for PCR based, HLA-DR subtyping(carried out essentially as described by Bunce (1995) Tissue Antigen46:355). The amino acid sequences of the resultant set of HLA-DRsubtypes for each biopsy were used to construct a set of critical Lym-1binding residues. A rare lymphoma biopsy that was not reactive withLym-1 was used as a negative control, to confirm that the criticalresidues were not present in its HLA-DR alleles.

For the lymphoma biopsy study, ten Lym-1 reactive and one Lym-1unreactive frozen biopsied lymphoma samples were used for PCR basedHLA-DR subtyping. The biopsies had been previously screened for Lym-1reactivity by immunoperoxidase staining. A score of greater than 20%cells stained was used as the lower limit for the Lym-1 reactive group.To isolate DNA and RNA from this tumor tissue, about 10 mg of fixedfrozen tissue (stored at minus 70° C.) was removed and sectioned. Theresulting frozen pieces were pulverized with a Gibco stainless steelpulverized that had been precooled in liquid nitrogen for 5 min. TheGibco Triazol protocol (see, e.g., Sewall (1998) Gibco Focus Vol 20.2)was used to isolate the RNA, DNA, and protein from the tissue samples.

The nucleic acid isolated and purified from fresh frozen lymphoma tumortissue biopsies was amplified by polymerase chain reaction (PCR).Biotinylated primers were used to amplify exon 2 of the Class II DRB2,DRB3, DRB4, and DRB5 genes. This subtyping system is based on thehybridization of biotinylated PCR products from exon 2 with immobilizedsequence specific oligonucleotides. The primers and detection systemwere purchased as a kit (Inna-Lipa system, Murex Diagnostics Inc.,Norcross, Ga.) and used according to the manufacturer's instruction.

To analyze HL4-DR subtyping results, the amino acid sequences of allpossible HLA-DR alleles indicated by the DR subtyping analysis of eachbiopsy were treated as a set and the HLA-DR subtypes contained in thesesequences were compared to the critical binding residues that arepresent in Lym-1 reactive HLA-DR10 beta chain, but absent in thesequences of Lym-1 unreactive DR3 and DR52 beta chains.

Only 1 of the 11 lymphoma biopsy samples was homozygous for a singleHLA-DR. Interestingly, none of the eleven biopsy tissues expressed therare HLA-DR10 allele (the original immunogen), while seven of the tenLym-1 reactive patients expressed DR53.

The amino acid sequence of Q70 or R70 followed by R71 (Q/R70-R71) wasfound in all 10 of the Lym-1 reactive samples and was absent in the oneLym-1 unreactive biopsy. The Lym-1 unreactive biopsy was homozygous forHLA-DR1(0103). In summary, all of the Lym-1 reactive biopsies expressedat least one HLA-DR allele that contained the following set of putativecritical Lym-1 binding residues: Q/R70, R71, A73, T77, and G86, that arecontained in DR10 but not in DR3 or DR52. However A73, T77, and G86 areall contained in the alleles expressed by the Lym-1 unreactive biopsy,leaving only Q/R70-R71 as the common Lym-1 binding residues in thisbiopsy sampling.

Complement Dependent Cytotoxicity Assays

To confirm the results of the lymphoma biopsy study, complementdependent cytotoxicity assays were used to test a variety of naturallyoccurring substitutions of the critical Lym-1 binding residues (thesetests were conducted essentially as described by Brown, et. al., “HLA-DRand DQ Serotyping; in Handbook of HLA Typing Techniques, ed. Hiu, et.al, CRC Press). Cells expressing known HLA-DR subtypes were assayed forLym-1 reactivity. The complement dependent cytotoxicity assays included37 lymphoblastoid cell lines and peripheral blood lymphocytes (PBL) from35 healthy donors and 50 chronic lymphocytic leukemia (CLL) patients.

For the complement dependent cytotoxicity assay of lymphoblastoid celllines, a total of 37 lymphoblastoid cell lines were assayed. Theseincluded 11 heterozygous lines and 26 homozygous lines, all of whichwere known to express certain HLA-DR alleles. Cell lines were assayed atconcentrations of Lym-1 mAb ranging from 0.1 mg/ml to about 0.1 ug/mL.This corresponds to a concentration range of approximately 0.67 uM to0.65 nM of Lym-1 mAb.

Complement dependent cytotoxicity assays were also conducted on freshlymphocyte isolates. Peripheral blood lymphocytes (PBL) from healthydonors and from chronic lymphocytic leukemia (CLL) patients wereisolated. A total of 85 samples of PBLs were assayed including 50samples from CLL patients and 35 from healthy donors. PBLs were assayedat concentrations of Lym-1 mAb ranging from 0.1 mg/ml to about 6 ug/ml,which corresponds to a concentration range of approximately 0.67 uM to42 nM of Lym-1 mAb. Lower concentrations were not used as the PBLs fromboth normal donors and CLL patients are much less reactive thanlymphoblastoid cell lines.

For the cytotoxicity test scoring, each dilution was assessed by eye andgiven a score of 1, 2, 4, 6, or 8, as is the normal convention in HLAserotyping cytotoxicity assays (see Brown, et al., Handbook of HLATyping Techniques, supra). The value “1” was assigned to correspond to0-10% cell death, “2” was assigned to correspond to 10-20% cell death,“4” was assigned to correspond to 20-30% cell death, 6 was assigned tocorrespond to 30-80% cell death, and 8 was assigned to correspond to80-100% cell death. The mean of each range was used as the percent celldeath corresponding to that score. A score of 1 is taken to indicate 5%cell death (the mean of the 0-10% range), a score of 2 is taken toindicate 15% cell death (the mean of the 10-20% range), 4 is taken toindicate 25% (the mean of the 20-30% range), 6 indicates 55% cell death(the mean of the 30-80% range), 8 indicates 90% cell death (the mean ofthe 80-100% range). All scores (translated to mean percentage) wereplotted (on a y axis) against a multiple of the log of the mAbconcentration (on the x axis). A sigmoid dose response curve resultedfor each dilution series. The limits of 1 and 8 are usual scores forseveral extremely high or low dilutions in the series. From this sigmoidcurve the Lethal Dose that killed 50% (LD50) of the cells wasinterpolated.

To convert LD50 to K_(d) and delta G (change in free energy) of avidity,the equilibrium equation (A+B)<=>(A·B) can be arranged to give thedissociation constant K_(d)=[A]*[B]/[A′B]. When [B] is equal to [A·B],50% of the antigens are bound by the mAb and [B]/[A·B] cancels outleaving K_(d)=[A]50. The concentration of mAb at LD50 can then beconverted to a free energy of binding by use of the equation deltaG=−RT1nK. The values can be expressed in kilojoules/mole (kJ/mol) orkilocalories/mole (kCal/mol). It is assumed that the lymphoblastoid celllines used here have comparable amounts of cell surface antigen andcytotoxicity response. Delta G refers to avidity rather than affinityhere, because each mAb has two binding sites. The cell lines were rankedfrom highest to lowest avidity based on LD50 data. The ranking wasanalyzed using StatXact.3 software (Cytel Software Corp., Cambridge,Mass.)

This analysis of the complement dependent cytotoxicity assaysdemonstrated that (in results similar to the biopsy DR beta chainstructural analysis) the only region that is, in this sampling of cells,always present in the reactive alleles and almost always absent in theunreactive alleles is Q/R70-R71. Surprisingly, in addition to Q/R70-R71,V85 also appears to be necessary for Lym-1 binding, as demonstrated bylow the avidity of HLA-DR102.

In summary, fifty chronic lymphocytic leukemia (CLL) cell samples and 35peripheral blood lymphocytes (PBL) samples from healthy donors weresubtyped for HLA-DR alleles. They were also tested for Lym-1 reactivity.Lym-1 reacted similarly with PBLs and CLLS, demonstrating much loweravidities for both healthy PBLs, and CLLS, than for the lymphoblastoidcell lines tested. Lym-1 was completely unreactive with approximately75% of the PBL and CLL samples tested (64 out of 85). Lym-1 showed thegreatest avidity for three PBL samples (1.8×10⁸) and one CLL cell line(1.1×10⁸) and very weak avidity (0.1 to 0.5×10⁸) for 17 other PBLsamples and CLL cell lines. 20 of the 21 reactive cells expressed HLA-DRalleles containing Q/R70-R71 and V85. However, about half of the 64unreactive PBLs and CLLs tested also contained these residues. Itappears from these results that the critical Lym-1 binding residues arenecessary for Lym-1 binding but that a second parameter, perhaps athreshold antigen density, is also important.

Summary

The Lym-1 reactive patient data demonstrates that, for the cells tested,critical Lym-1 binding residues are Q/R70-R71, A73, T77, and G86. Theseare the residues that are included in the set of the differences betweenthe amino acid sequences of (Lym-1 reactive) DR10 and (Lym-1 unreactive)DR3 & DR52. These residues are also the amino acid sequences of thehaplotypes of all ten Lym-1 reactive lymphoma biopsies tested. However,the Lym-1 unreactive biopsy contained all of the putative critical Lym-1binding residues except Q/R70-R71. This demonstrates that the presenceof A73, T77, and G86 is not sufficient for Lym-1 binding and that thesequence Q/R70-R71 is critical for Lym-1 binding to the HLA-DR allelestested.

To confirm these results, Lym-1 was tested with a number of homozygousand heterozygous cell lines in complement dependent cytotoxicity assays.Thirty-seven lympho-blastoid cell lines, including thirty-one HLA-DRgenotypes, were screened for Lym-1 reactivity. The results confirmedthat all of the strongly reactive cells expressed at least oneQ/R70-R71-containing HLA-DR allele, while none of the least reactivecell lines expressed that sequence at positions 70 to 71 of the DR betachain. Cell lines containing the DR53 allele were always reactive. DR53beta chains (DRB4-01011 and 0103) and DR10 beta chain (DRB1-1001)contain identical sequences between positions 49 to 73.

One false positive and one false negative were found in the weaklyreactive categories. The results were ranked and statistically analyzedusing StatXact.3 software. This analysis tests the significance ofranked data and the probability that the ranking could have resulted bychance. The simple hypothesis, based on biopsy study results, whichstates that the presence of Q/R70-R71 is critical for Lym-1 binding toHLA-DR was tested. The null hypothesis, which states that thecytotoxicity assay ranking could have occurred by chance, was rejectedat a confidence level>>0.999.

An anomaly was found with HLA-DRB1*0102 (DR102). DR102 contains thecritical Q/R70-R71 sequence, but it is not strongly reactive with Lym-1.DR102 is identical to the highly reactive DRB1*0101 (DR101) with theexception of residues 85 and 86. In DR102 the V85-G86 of DR101 has beenreplaced by A85-V86. This difference is apparently enough to reduce theavidity of Lym-1 for DR102. It should be noted that two other of theweakest binding cell types also express alleles that contain the A85-V86replacement, while none of the strongest binders contain A85-V86.However, V86 is a common substitution found in both strong and weakLym-1 binding alleles and is therefore ruled out as a critical residue,leaving V85 as a critical Lym-1 binding residue.

The sequences of the HLA-DR alleles with highest and lowest Lym-1avidity were compared. Three residues, Q/R70, R71 and V85, are found inall of the high avidity alleles and none of the low avidity alleles. K71appears in the sequence of the DR4 allele; however, it could not beverified whether this Q70-K71 sequence is reactive since the highlyreactive DR53 allele, which contains R70-R71, is co-expressed withHLA-DR4 in all the cell lines tested. Residues Q70-K71 also appears inthe sequences of several low avidity alleles, particularly, DR3 andDR52.

Therefore, for the cells tested, critical residues for strong Lym-1binding to the HLA-DR beta chain are Q/R70-R71 and V85. These residuesappear solvent exposed and oriented on the same face of the alpha helixof HLA-DR beta chain (see FIG. 1, schematically illustrating criticalLym-1 binding residues on HLA-DRI, the three dimensional structure fromthe Brookhaven Protein Data Bank (available, e.g., athttp://pdb.pdb.bnl.gov/) file of the HLA-DR crystal structure by Brown(1993) supra. The DR beta sequence D70-R71 is unreactive, D70 apparentlynegating the critical Lym-1 binding residue R71. The sequence D70-E71 isunreactive, as might be expected because of the double negative charge.Alleles DR15 and 16 display Q70-A71, a net neutral charge, and show nodetectable Lym-1 binding.

To summarize, a net neutral (Q70-A71, D70-R71) or negative charge(D70-E71) at residues 70-71 drastically reduces Lym-1 binding. A netpositive charge (Q70-R71, R70-R71) at these positions appears to becritical for Lym-1 binding. Thus, the Lym-1 epitope can present anegative or neutral residue to the Class II molecule's antigen bindingpocket.

The avidity constants measured here of Lym-1 with various cell lines arein general agreement with Epstein's avidity calculation for Lym-1binding to Raji (Epstein (1987) supra). They found that Lym-1 bindspreferentially to lymphoblastoid cells with an avidity of 4.0×10⁸, whichis 5-fold greater than the avidity of 0.8×10⁸ they found for Lym-1binding to healthy tonsil B-cells. Results from the instant studies showthe top 16 Lym-1 binding cell lines ranged in avidity from a maximum ofapproximately 7.4×10⁸ (DR804/9/53 and DR10/1202/52), to minimum of about0.5×10⁸. Of this group, 14 cell lines express at least one allele thatcontains the residues Q/R70-R71 and V85. In contrast only 1 of the 21genotypes with avidity of less than about 0.5×10⁸ has an allele thatcontains the critical residues (a false negative).

Lymphoblastoid cell lines are known to express much higher surfaceantigen density than normal peripheral blood cells. These results arealso in general agreement with results from Epstein's studies (Epstein(1987) supra), which showed Lym-1 binding selectively to lymphomas ascompared to normal healthy tonsil B-cells. The results from the instantcytotoxicity tests of Lym-1 with PBLs from healthy donors and CLLpatients showed that these cells were much less reactive, regardless ofwhether they contained the Q/R70-R71 & V85 epitope. This may infer thatalthough the critical Lym-1 binding residues are necessary, cell surfaceantigen density may also be useful in assessing for clinically usefulavidity.

2 1 16 PRT Artificial Sequence Description of ArtificialSequencesubsequence of Class II major histocompatability molecule thatgenerates an immune response to non-Hodgkin′s B cell lymphoma cell 1 XaaArg Arg Xaa Ala Xaa Xaa Thr Xaa Cys Xaa Xaa Xaa Xaa Xaa Val 1 5 10 15 216 PRT Artificial Sequence Description of Artificial Sequencesubsequenceof Class II major histocompatability molecule that generates an immuneresponse to non-Hodgkin′s B cell lymphoma cell 2 Xaa Arg Arg Ala Ala ValAsp Thr Tyr Cys Arg His Asn Tyr Gly Val 1 5 10 15

What is claimed is:
 1. An isolated peptide consisting of a subsequenceof a Class II major histocompatibility molecule, wherein the peptide hasthe following properties, (a) having a structure comprisingR₁-R₂-R₃-R₄-R₅-R₆-R₇-R₈-R₉-R₁₀-R₁₁-R₁₂-R₁₃-R₁₄-R₁₅-R₁₆, wherein R₁ isGln, Lys, or Arg; R₂ is Arg; R₃ and R₄ are members independentlyselected from the group consisting of all amino acids; R₅ is Ala, Glu,Asp, Val, Leu or Ile; R₆ and R₇ are members independently selected fromthe group consisting of all amino acids; R₈ is Thr; R₉, R₁₀, R₁₁, R₁₂,R₁₃, R₁₄, and R₁₅ are members independently selected from the groupconsisting of all amino acids; and, R₁₆ is Val; (b) capable ofgenerating an immune response to a non-Hodgkin's B cell lymphoma cell.2. The isolated peptide of claim 1, wherein R₁ is Gln, Lys, or Arg; R₂is Arg; R₃ is Arg; R₄ is selected from the group consisting of all aminoacids; R₅ is Ala; R₆ and R₇ are members independently selected from thegroup consisting of all amino acids; R₈ is Thr; R₉ is selected from thegroup consisting of all amino acids; R₁₀ is Cys; R₁₁, R₁₂, R₁₃, R₁₄, andR₁₅ are members independently selected from the group consisting of allamino acids; and, R₁₆ is Val (SEQ ID NO: 1).
 3. The isolated peptide ofclaim 2, wherein R₁ is Gln, Lys, or Arg; R₂ is Arg; R₃ is Arg; R₄ isAla; R₅ is Ala; R₆ is Val; R₇ is Asp; R₈ is Thr; R₉ is Tyr; R₁₀ is Cys;R₁₁ is Arg; R₁₂ is His; R₁₃ is Asn; R₁₄ is Tyr; R₁₅ is Gly, and R₁₆ isVal (SEQ ID NO: 2).
 4. The isolated peptide of claim 1, furthercomprising a pharmaceutically acceptable excipient.
 5. A compositioncomprising an adjuvant and the isolated peptide of claim
 1. 6. Theisolated peptide of claim 1, wherein the non-Hodgkin's lymphoma cell isselected from the group consisting of a B-cell chronic lymphocyticleukemia/small lymphocytic lymphoma (B-CCL/SLL) cell, alymphoplasmacytoid lymphoma (LPL) cell, a follicular lymphoma (FL) cell,a mucosa-associated lymphoid tissue lymphoma (MALTL) cell, a spleniclymphoma with villous lymphocytes (SLVL) cell and a mantle cell lymphomacell.
 7. A method for generating an antibody reactive with anon-Hodgkin's B cell lymphoma cell, comprising administering animmunogenically effective amount of a isolated peptide of claim 1 to amammal.
 8. The method of claim 7, wherein R₁ is Gln, Lys, or Arg; R₂ isArg; R₃ is Arg; R₄ is Ala; R₅ is Ala; R₆ is Val; R₇ is Asp; R₈ is Thr;R₉ is Tyr; R₁₀ is Cys; R₁₁ is Arg; R₁₂ is His; R₁₃ is Asn; R₁₄ is Tyr;R₁₅ is Gly, and R₁₆ is Val (SEQ ID NO: 2).
 9. The method of claim 7,wherein the non-Hodgkin's lymphoma cell is selected from the groupconsisting of a B-cell chronic lymphocytic leukemia/small lymphocyticlymphoma (B-CCL/SLL) cell, a lymphoplasmacytoid lymphoma (LPL) cell, afollicular lymphoma (FL) cell, a mucosa-associated lymphoid tissuelymphoma (MALTL) cell, a splenic lymphoma with villous lymphocytes(SLVL) cell and a mantle cell lymphoma cell.
 10. A method of inducing animmunogenic response directed to a polypeptide epitope, comprisingadministering an immunogenically effective amount of a compositioncomprising a polypeptide epitope to a mammal, wherein the epitopecomprises an amino acid sequence having a structure comprisingR₁-R₂-R₃-R₄-R₅-R₆-R₇-R₈-R₉-R₁₀-R₁₁-R₁₂-R₁₃ -R₁₄-R₁₅-R₁₆, wherein R₁ isGln, Lys, or Arg; R₂ is Arg; R₃ and R₄ are members independentlyselected from the group consisting of all amino acids; R₅ is Ala, Glu,Asp, Val, Leu or Ile; R₆ and R₇ are members independently selected fromthe group consisting of all amino acids; R₈ is Thr; R₉, R₁₀, R₁₁, R₁₂,R₁₃, R₁₄, and R₁₅ are members independently selected from the groupconsisting of all amino acids; and, R₁₆ is Val.
 11. The method of claim10, wherein R₁ is Gln, Lys, or Arg; R₂ is Arg; R₃ is Arg; R₄ is Ala; R₅is Ala; R₆ is Val; R₇ is Asp; R₈ is Thr; R₉ is Tyr; R₁₀ is Cys; R₁₁ isArg; R₁₂ is His; R₁₃ is Asn; R₁₄ is Tyr; R₁₅ is Gly, and R₁₆ is Val (SEQID NO: 2).
 12. The immunogenic method of claim 10, wherein theimmunogenic response generates antibodies specific for the polypeptideepitope.
 13. The method of claim 10, wherein the mammal is a human, amouse or a rabbit.